Adiponectin Gene Polymorphism and Ischemic Stroke Subtypes in a Chinese Population

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Adiponectin Gene Polymorphism and Ischemic Stroke Subtypes in a Chinese Population Shanshan Li,* Ning Lu,* Zhongnan Li,† Bin Jiao,* Hanping Wang,‡ Jia Yang,§ and Tao Yu†

As an adipose tissue-specific protein, adiponectin has been suggested as a protective factor for stroke, acting through anti-inflammatory and antiatherogenic effects. Therefore, we aimed to investigate whether 3 polymorphisms (rs1501299, rs2241767, and rs3774261) in the adiponectin (ADIPOQ) gene and their haplotypes were associated with ischemic stroke (IS) and its subtypes in a Chinese population. ADIPOQ gene rs1501299, rs2241767, and rs3774261 polymorphisms were analyzed in 385 IS patients, including 182 patients with large-artery atherosclerosis (LAA), 203 patients with small-vessel occlusion (SVO), and 418 matched controls. The subjects were genotyped by using polymerase chain reaction–restriction fragment length polymorphism analysis. In univariate logistic analysis, the A allele frequency of rs2241767 was moderately higher in IS and LAA patients than that in controls (P = .028 and P = .017, respectively). Compared with the wide-type AA homozygote, both the genotype GG and the dominant model (GG+AG) of rs2241767 moderately increased the risk of LAA (P = .040 and P = .034, respectively). In multivariate logistic regression analysis, the genotype GG of rs2241767 was independently related to IS and LAA patients (adjusted, odds ratio [OR] = 1.822, 95% confidence interval [CI]: 1.037-3.202, P = .037 and OR = 2.051, 95% CI: 1.041-4.041, P = .038, respectively) rather than SVO. In contrast, no relationship was observed between the polymorphism of rs1501299 and rs3774261 and either subtype of IS using both univariate and multivariate logistic regression analyses. In addition, the Trs1501299Grs2241767-Ars3774261 haplotype showed a moderately increased risk of IS and LAA (OR = 1.595, 95% CI: 1.058-2.406, P = .025 and OR = 1.709, 95% CI: 1.047-2.789, P = .031, respectively) but not of SVO. In conclusion, this study tentatively demonstrated that the polymorphism of rs2241767 and the Trs1501299-Grs2241767-Ars3774261 haplotype were associated with susceptibility to IS and LAA in a Chinese population. Key Words: Adiponectin—ADIPOQ gene—polymorphism—ischemic stroke—subtypes. © 2016 National Stroke Association. Published by Elsevier Inc. All rights reserved.

Introduction From the *Department of Emergency, Huludao Center Hospital, Huludao, Liaoning, China; †Division of Radiation Imaging, Huludao Center Hospital, Huludao, Liaoning, China; ‡Department of Cardiology, Huludao Center Hospital, Huludao, Liaoning, China; and §Intensive Care Unit, Huludao Center Hospital, Huludao, Liaoning, China. Received August 23, 2016; revision received October 19, 2016; accepted October 31, 2016. Address correspondence to Tao Yu, Division of Radiation Imaging, Huludao Center Hospital, No. 15 Lianshan Street, Lianshan District, Huludao, Liaoning, China. E-mail: [email protected]. 1052-3057/$ - see front matter © 2016 National Stroke Association. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2016.10.045

Stroke is the leading cause of death and one of the most common causes of disability in China.1 About 80% of all strokes are ischemic stroke (IS).2 As a multifactorial disease, the etiology of IS is complex, involving both genetic and environmental factors.3 Studies on genetic factors predisposing to IS have been repeatedly reported.4-6 In addition, inflammation-related atherosclerosis plays an essential role in the pathogenesis of IS.7,8 Therefore, genes involved in inflammatory responses are under investigation to identify variant susceptibility to IS. As an adipose tissue-specific protein, adiponectin plays a vital role in modulating lipid and glucose

Journal of Stroke and Cerebrovascular Diseases, Vol. ■■, No. ■■ (■■), 2016: pp ■■–■■

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metabolism, inhibition of vascular smooth muscle cell proliferation, and antiatherogenic and anti-inflammatory activities in the vascular system.9-11 Various reports have indicated that lower circulating levels of adiponectin are associated with higher risk of metabolic syndrome,12 type 2 diabetes (T2DM),13 myocardial infarction,14 and IS.15,16 The adiponectin gene, ADIPOQ, is located on chromosome locus 3q27, and contains 3 exons and 2 introns and spans 16 kb.17 A family study shows that adiponectin levels are influenced by genetic factors, with heritability in the range of 30%-50%.18 Several reports have shown an association between genotypes within the ADIPOQ gene and adiponectin levels.19-21 Associations between ADIPOQ gene polymorphisms and IS also have been reported in different populations; however, current results have been controversial.22-24 Phenotypic and genetic heterogeneities may be attributed to these inconsistent results. Heritability for IS varied for different stroke subtypes,4 and ignoring etiological differences might give rise to inconsistent conclusions.25 Therefore, we performed this case– control study to investigate whether 3 SNPs (rs1501299, rs2241767, and rs3774261) in the ADIPOQ gene were associated with the risk of IS and subtypes in a Chinese Han population.

Materials and Methods Study Population In the present study, a total of 556 unrelated consecutive patients were recruited from the Department of Neurology of Huludao Center Hospital from January 2011 to June 2014. IS was diagnosed with neurological examination and computed tomography or magnetic resonance imaging scan confirming the presence of infarction within 7 days of onset. IS subtype was classified by 2 neurologists according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification26: large-artery atherosclerosis (LAA, 182 subjects), small-vessel occlusion (SVO, 203 subjects), cardioembolism (71 subjects), the other demonstrated etiology (58 subjects), and undetermined etiology (42 subjects). Because of small samples in the cardioembolic stroke and other demonstrated etiology and undetermined etiology subgroups, these 3 subgroups had been excluded from etiological classification. A total of 418 age- and gender-matched persons without IS were recruited from Aerospace Center Hospital Medical Center as the control group during the same period. Patients who had trauma, blood diseases, tumors, serious chronic diseases, severe inflammatory diseases, and autoimmune diseases were all excluded. Informed consent was obtained from all participants enrolled in the present study. The study protocol was approved by the Human Ethics Committee of Huludao Center Hospital.

Laboratory Methods Peripheral venous blood samples were drawn from all subjects for measurement of lipoprotein after an overnight fast. Plasma concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDLC), and high-density lipoprotein cholesterol (HDL-C) were determined by an automatic biochemistry analyzer (Hitachi 7100, Hitachi Limited, Tokyo, Japan).

Collection of Clinical Data A standardized questionnaire was generated to collect information from all participants. Information was obtained either directly from the patient or, if the participant was unable to communicate, a close relative. Subjects with a systolic blood pressure of 140 mmHg or higher or a diastolic blood pressure of 90 mmHg or higher on at least 2 separate occasions, or were chronically (>6 months from evaluation) taking antihypertensive drugs were defined as hypertensives. Subjects with fasting glucose levels of 7.0 mmol/L or higher or glucose levels of 11.1 mmol/L or higher or were taking hypoglycemic drugs were considered diabetic. Subjects who have at least one of high TC (≥6.21 mmol/L), high LDL-C (≥4.16 mmol/L), low HDL-C (<1.03 mmol/L), and high TG (≥2.26 mmol/L) were considered dyslipidemic. Smoking habits were classified as current, former, and never smokers; current smokers were those who were currently smoking at presentation or had stopped within the past 6 months; former smokers were individuals who had stopped for more than 6 months before presentation; never smokers were characterized as individuals who had never smoked previously; former and never smokers were classified as nonsmokers for statistical analysis. Drinkers were individuals who consume 70 g of alcohol or higher per week for more than 1 year. Body mass index (BMI) was calculated by body weight in kilogram divided by height in meter squared.

Single-Nucleotide Polymorphism (SNP) Selection The ADIPOQ gene is located on chromosome locus 3q27, and contains 3 exons and 2 introns and spans 16 kb. Haplotype-tagging SNPs of the ADIPOQ gene were chosen according to the publicly available HapMap CHB databank (http://hapmap.ncbi.nlm.nih.gov/). To identify common haplotype-tagging SNPs, the eligible SNPs were selected using the Tagger program in HaploView software (version 4.2) (https://www.broadinstitute.org/ haploview/haploview). We searched the SNPs with a minor allele frequency of .05 or higher and an r2 value of .8 or higher, or those established by previous articles as candidate SNPs. Finally, 3 SNPs were selected, including rs1501299, rs2241767, and rs3774261.

SNP Genotyping Genomic DNA was extracted from leukocytes in a peripheral blood sample with the salt fractionation method.

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Table 1. Primer sequences and reaction conditions for genotyping assay of the ADIPOQ gene Annealing temperature (°C)

Polymorphism

Primers sequences (5′-3′)

rs1501299 (+276G/T)

F: CCC GGA GTC CTG GTT CCC CTG CCC A R: TGT CTA GGC CTT GGT TAA TAA TGA A F: GGT GAG AAG GTG AGA AAG GAG R: TAC TGG GAA TAG GGA TGA GGG F: TGG CAT TCA CCC ACA TTT AC R: AAG CCT TCA GTC TTC ATC AG

rs2241767 (+349A/G) rs3774261 (+712G/A)

Restriction enzyme

Fragment length (bp)

55

Bsm I

62

Bsu36 I

59

Rsa I

231 206 + 25 292 163 + 129 217 183 + 34

Abbreviations: F, forward; R, reverse.

The primers of rs1501299, rs2241767, and rs3774261 were designed using Primer-BLAST software (https:// www.ncbi.nlm.nih.gov/tools/primer-blast/) and synthesized by Shanghai Invitrogen Biotechnology Co., Ltd., Shanghai, China. The genotyping was performed using polymerase chain reaction (PCR). The PCR included the following: total reaction volume, 20 μL, including template DNA, 10 ng; 2 × PCR buffer, 10 μL; Taq enzyme, 1.5 U; deoxynucleotide, .2 mmol/L; 2 primers of 1 μL each, in 94°C predenaturation for 5 minutes; 35 cycles of 94°C denaturation for 30 seconds; 60°C annealing for 30 seconds; and 72°C extension for 90 seconds, followed by 72°C extension for 10 minutes. The PCR primer sequences, the annealing temperature, the restriction enzyme, and PCR product sizes are shown in Table 1. A total of 10% of samples were regenotyped for each SNP at random to confirm the genotyping, and all results were consistent.

Statistical Analysis Continuous variables were expressed as the mean ± standard deviation. For normally distributed data, differences between the 2 study groups were determined using the Student t-test; for non-normal continuous data, the Mann– Whitney U-test was used. The χ2 test or the Fisher exact test was used not only to examine differences in categorical variables but also to evaluate the Hardy– Weinberg equilibrium and the differences in allele and genotype frequencies between cases and controls. In addition, IS and its subtypes associated with each genotype in this case–control analysis were evaluated by logistic regression analysis while controlling for age, gender, and the potential confounding factors. The odds ratios (ORs) (95% confidence interval [CI]) and corresponding P values were also calculated. All statistical analyses were performed with SPSS software (version 10.0; SPSS Inc., Chicago, IL). A P value less than .05 was considered significant (2-tailed). Analyses of the linkage disequilibrium (LD) and haplotype construction were performed using the online software SHEsis (http://analysis.bio-x.cn/ myAnalysis.php). A haplotype frequency below 3% was excluded from the analysis.

Results Characteristics of Subjects The baseline characteristics of the studied population are summarized in Table 2. The patients with stroke subtypes and controls were well matched for age and gender. In addition, the mean HDL-C and TC levels did not differ between stroke subtypes and controls. However, compared with controls, the frequencies of smoking, diabetes, hypertension, dyslipidemia, BMI, TG, and LDL-C levels were higher in either IS or subtype cases. The prevalence of drinking was more frequent among IS or LAA patients than controls rather than SVO.

ADIPOQ Genotypes and Their Risk The genotype, allele, and different genetic model frequencies of the SNPs of the ADIPOQ gene in patients stratified for stroke subtypes and control subjects are shown in Tables 3 and 4. The genotype distributions of rs1501299, rs2241767 and rs3774261 in controls were consistent with the Hardy–Weinberg equilibrium (χ2 = 2.162, P = .141; χ2 = .218, P = .640; χ2 = .436, P = .509, respectively). No significant difference was found in the distributions of genotypes of rs1501299, rs2241767, and rs3774261 between IS patients or subtypes of patients and controls. In contrast, the A allele frequency of rs2241767 was moderately higher in IS and LAA patients than that in controls (OR = 1.276, 95% CI: 1.027-1.586, P = .028; OR = 1.368, 95% CI: 1.060-1.812, P = .017, respectively). In addition, compared with the wide-type AA homozygote, both the genotype GG and the dominant model (GG+AG) of rs2241767 moderately increased the risk of LAA (OR = 1.441, 95% CI: 1.016-2.044, P = .040; OR = 1.925, 95% CI: 1.045-3.546, P = .034, respectively). However, the polymorphism of rs2241767 was not associated with SVO. Furthermore, after adjustment for age, gender, smoking, alcohol drinking, BMI, diabetes, hypertension, TC, TG, HDL-C, and LDL-C by multivariate logistic regression analysis, the genotype GG of rs2241767 was independently related to IS and LAA patients (adjusted, OR = 1.822, 95% CI: 1.037-3.202, P = .037; OR = 2.051, 95% CI:

ARTICLE IN PRESS S. LI ET AL. Abbreviations: BMI, body mass index; HDL-C, high-density lipoprotein cholesterol; IS, ischemic stroke; LAA, large-artery atherosclerosis; LDL-C, low-density lipoprotein cholesterol; SVO, small-vessel occlusion; TC, total cholesterol; TG, triglyceride.

.624 .250 .013 .158 .002 <.000 <.000 .020 .457 .004 .004 .283 .213 .432 .007 .012 .001 <.000 <.000 .002 .188 <.000 <.000 .063 61.65 ± 12.14 227 (59.0) 111 (28.8) 79 (20.5) 24.75 ± 2.81 113 (29.4) 244 (63.4) 147 (38.2) 4.63 ± 1.25 1.48 ± .68 2.75 ± .72 1.27 ± .35 60.69 ± 10.59 235 (56.2) 86 (20.6) 58 (13.9) 24.12 ± 2.68 62 (14.8) 158 (37.8) 116 (27.8) 4.53 ± 1.10 1.33 ± .62 2.60 ± .68 1.32 ± .38 Age (years) Male, n (%) Smoking, n (%) Alcohol drinking, n (%) BMI (kg/m2) Diabetes, n (%) Hypertension, n (%) Dyslipidemia TC (mmol/L) TG (mmol/L) LDL-C (mmol/L) HDL-C (mmol/L)

62.21 ± 12.58 103 (56.6) 51 (28.0) 42 (23.1) 24.64 ± 2.93 52 (28.6) 115 (63.2) 72 (39.6) 4.67 ± 1.25 1.50 ± .69 2.80 ± .68 1.26 ± .27

.152 .932 .046 .005 .034 <.000 <.000 .004 .143 .001 .001 .073

61.15 ± 11.74 124 (61.1) 60 (29.6) 37 (18.2) 24.85 ± 2.71 61 (30.0) 129 (63.5) 75 (36.9) 4.60 ± 1.25 1.46 ± .67 2.77 ± .73 1.28 ± .41

P value P value P value Controls (n = 418) Characteristic

Patients with LAA (n = 182) All patients with IS (n = 385)

Table 2. Baseline characteristics of the study population

Patients with SVO (n = 203)

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1.041-4.041, P = .038, respectively). In contrast, no relationship was observed between the polymorphism of rs1501299 and rs3774261 and either subtype of IS using both univariate and multivariate logistic regression analyses.

Haplotype Analysis LD analysis showed that rs1501299, rs2241767, and rs3774261 SNPs had weak LD in the present study population according to the results generated from the SHEsis software. In the haplotype analysis, 8 haplotypes were identified, of which the Trs1501299-Grs2241767-Grs3774261 haplotype, with a frequency below 3%, was excluded. As shown in Table 5, the Trs1501299-Grs2241767-Ars3774261 haplotype showed a moderately increased risk of IS and LAA (OR = 1.595, 95% CI: 1.058-2.406, P = .025; OR = 1.709, 95% CI: 1.0472.789, P = .031, respectively) rather than SVO.

Discussion In the present case–control investigation, 3 SNPs of the ADIPOQ gene (rs1501299, rs2241767, and rs3774261) were examined for their association with IS and its subtypes in a Chinese Han population. The results of our study revealed that the SNP rs2241767 and the haplotype G-A-G (comprising rs1501299, rs2241767, and rs3774261) were moderately associated with the increased risk of LAA subtype instead of the SVO subtype. However, no relationship was found between the SNPs rs1501299 and rs3774261 and the risk of IS and its subtypes. Several reports have shown that genetic variation in the ADIPOQ gene may contribute to atherosclerosis27 and inflammation,28 all of which are associated with the development of IS. However, studies on the relationship between ADIPOQ gene variations and the risk of IS and its subtypes are still infrequent and inconsistent. As for rs2241767, this is the first study to our knowledge to reveal the association between rs2241767 and IS and its subtypes. Our results showed a modest association of the G allele carriers with increased risk of IS and LAA. The significant differences remained after adjustment for potential confounders (including age, gender, smoking, alcohol drinking, BMI, diabetes, hypertension, TC, TG, HDL-C, and LDL-C). A recent study by Wassel et al 29 demonstrated that the genotypes AG/GG of rs2241767 had 36% greater (95% CI: 16-59, P = .0001) coronary artery calcium prevalence and also had a larger common carotid intima media thickness (P = .0043) among African–American but not in Chinese or Caucasian participants. The molecular mechanism of the association between rs2241767 and IS is still unclear. It is interesting to find that rs2241767 is located in intron 2, which is a noncoding region of a gene. However, evidence shows that introns can significantly affect gene expression such as contain enhancer elements or elevate mRNA accumulation.30,31 Gu32 found that the ADIPOQ gene has a moderate LD in the

rs1501299

Genotype

Allele rs2241767

Genotype

Allele rs3774261

Genotype

Allele

GG GT TT G T AA AG GG A G AA AG GG A G

All patients with IS (n = 385)

Patients with LAA (n = 182)

Patients with SVO (n = 203)

Number (%)

Number (%)

P value

Number (%)

P value

Number (%)

P value

220 (52.6) 158 (37.8) 40 (9.6) 598 (71.5) 238 (28.5) 231 (55.3) 157 (37.5) 30 (7.2) 619 (74.0) 217 (26.0) 136 (32.5) 199 (47.6) 83 (19.9) 471 (56.3) 365 (43.7)

193 (50.1) 145 (37.7) 47 (12.2) 531 (69.0) 239 (31.0) 187 (48.6) 158 (41.0) 40 (10.4) 532 (69.1) 238 (30.9) 119 (30.9) 204 (53.0) 62 (16.1) 442 (57.4) 328 (42.6)

.465

92 (50.5) 70 (38.5) 20 (11.0) 254 (69.8) 110 (30.2) 84 (46.2) 77 (42.3) 21 (11.5) 245 (67.3) 119 (32.7) 55 (30.2) 99 (54.4) 28 (15.4) 209 (57.4) 155 (42.6)

.829

101 (49.8) 75 (36.9) 27 (13.3) 277 (68.2) 129 (31.8) 103 (50.7) 81 (39.9) 19 (9.4) 287 (70.7) 119 (29.3) 64 (31.5) 105 (51.7) 34 (16.8) 233 (57.4) 173 (42.6)

.366

.260 .095

.028 .236

.667

.539 .062

.017 .252

.729

.231 .463

.212 .544

.726

Abbreviations: IS, ischemic stroke; LAA, large-artery atherosclerosis; SVO, small-vessel occlusion.

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Controls (n = 418)

ADIPONECTIN GENE POLYMORPHISM AND ISCHEMIC STROKE SUBTYPES

Table 3. The genotype and allele frequencies of rs1501299, rs2241767, and rs3774261

5

6

Table 4. The different genetic model frequencies of rs1501299, rs2241767, and rs3774261

rs1501299

Recessive Dominant Additive

rs2241767

Recessive Dominant Additive

rs3774261

Recessive Dominant Additive

(GG+GT)/TT (TT+GT)/GG GT/GG TT/GG (AA+AG)/GG (GG+AG)/AA AG/AA GG/AA (AA+GA)/GG (GG+GA)/AA GA/AA GG/AA

Patients with LAA (n = 182)

Patients with SVO (n = 203)

Crude P value

Adjusted P value*

Crude P value

Adjusted P value

Crude P value

Adjusted P value*

.229 .479 .766 .216 .107 .058 .145 .054 .167 .621 .323 .450

.386 .332 .481 .306 .063 .094 .250 .037 .871 .330 .211 .990

.594 .693 .761 .552 .078 .040 .112 .034 .195 .576 .304 .503

.896 .444 .383 .919 .065 .110 .274 .038 .315 .328 .173 .793

.160 .501 .856 .162 .344 .289 .420 .265 .353 .801 .555 .399

.200 .405 .689 .179 .197 .237 .401 .146 .778 .529 .449 .915

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Genetic model

All patients with IS (n = 385)

Abbreviations: BMI, body mass index; HDL-C, high-density lipoprotein cholesterol; IS, ischemic stroke; LAA, large-artery atherosclerosis; LDL-C, low-density lipoprotein cholesterol; SVO, small-vessel occlusion; TC, total cholesterol; TG, triglyceride. *Adjusted for age, gender, smoking, alcohol drinking, BMI, diabetes, hypertension, TC, TG, HDL-C, and LDL-C.

S. LI ET AL.

ARTICLE IN PRESS .277 .419 .501 .929 .865 .439 .071 .860 (.655-1.129) .890 (.671-1.181) 1.125 (.798-1.588) .979 (.615-1.559) .968 (.663-1.413) 1.154 (.803-1.658) 1.555 (.959-2.253) Abbreviations: CI, confidence interval; IS, ischemic stroke; LAA, large-artery atherosclerosis; OR, odds ratio; SVO, small-vessel occlusion. *Haplotypes are listed as rs1501299, rs2241767, and rs3774261.

100 (24.6) 91 (22.4) 58 (14.3) 28 (7.0) 44 (11.0) 52 (12.7) 30 (7.5) .282 .647 .713 .187 .797 .190 .031 .855 (.643-1.137) .934 (.698-1.251) 1.071 (.745-1.539) 1.347 (.865-2.099) 1.052 (.715-1.548) .753 (.492-1.152) 1.709 (1.047-2.789) .282 .647 .713 .187 .797 .190 .025 G-A-A G-A-G G-G-A G-G-G T-A-A T-A-G T-G-A

229 (27.4) 203 (24.3) 107 (12.8) 59 (7.1) 94 (11.2) 94 (11.2) 41 (4.9)

186 (24.1) 179 (23.2) 110 (14.3) 56 (7.3) 88 (11.4) 80 (10.4) 59 (7.7)

.849 (.678-1.063) .948 (.753-1.195) 1.140 (.856-1.519) 1.051 (.719-1.535) 1.024 (.751-1.395) .925 (.674-1.270) 1.595 (1.058-2.406)

88 (24.2) 83 (22.8) 49 (13.5) 33 (9.1) 42 (11.5) 31 (8.5) 29 (8.0)

OR (95% CI) n (%) P value Haplotype*

n (%)

n (%)

OR (95% CI)

n (%)

OR (95% CI)

P value

Patients with SVO (n = 203) Patients with LAA (n = 182) All patients with IS (n = 385) Controls (n = 418)

Table 5. Haplotype frequency distribution of the ADIPOQ gene between IS and subtype patients and controls

P value

ADIPONECTIN GENE POLYMORPHISM AND ISCHEMIC STROKE SUBTYPES

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boundary of exon 2–intron 2. It is plausible that the positive association between rs2241767 and LAA occurs through LD with the functional of 1 polymorphism in exon 2. Besides, Du et al33 had reported that patients with the rs2241767 AG/GG genotype had lower levels of serum adiponectin (P = .044) than those with genotype AA in a Chinese population, which may lead to susceptibility to LAA. As for rs1501299 and rs3774261, Liu et al34 found that there was no significant difference in the distributions of genotypes (P = .637) and alleles (P = .375) of rs1501299 between IS patients and controls in a Chinese population. Similarly, Hegener et al24 found no evidence for an association of the 1501299 with risk of IS in American men. It has been shown that the AA genotype of rs3774261 conferred a .65 times lower risk of T2DM in a South Indian population.21 In contrast, no evidence was found for a correlation between rs3774261 and T2DM, insulin resistance, and the metabolic syndrome in European–Australian individuals.35 In the present study, our results did not find any correlation of rs1501299 and rs3774261 with the risk of IS or subtypes in a Chinese population. There are also some shortcomings in our study. First, the relationship between genetic variants of adiponectin and the circulating adiponectin levels cannot be assessed because we did not measure the plasma adiponectin concentrations. Second, the role of single-gene mutation may be insufficient to cause diseases or may produce only a weak effect, and the IS pathogenesis is a complex process involving multiple genes and environmental risk factors. Therefore, gene–gene or gene–environment interactions may also contribute to the results obtained here. Finally, we adopted a case–control study with a small sample size, which may cause low test performance and possibly produce negative results.

Conclusion In conclusion, we found that the rs2241767 polymorphism and haplotype of G-A-G (comprising rs1501299, rs2241767, and rs3774261) might contribute to the susceptibility to LAA in a Chinese population. Further studies with larger sample size are also required to confirm our results in different ethnicities. Acknowledgment: We express our sincere thanks to the medical staff of Huludao Center Hospital who provided great support to this study.

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